Mutation and Microsatellite Instability (MSI) Affect the Differential Gene Expression of Folic Acid and 5-Flourouracil Metabolism-Related Genes in Colorectal Carcinoma.

The CpG Island Methylator Phenotype and Chromosomal Instability Are Inversely Correlated in Sporadic Colorectal Cancer

26 Background: Microsatellite instable (MSI) metastatic colorectal cancer (CRC) tumors have been found to be responsive to immune checkpoint inhibitors. Most MSI tumors are in consensus molecular subtype CMS1. Here, in a search for microsatellite stable (MSS) samples that may respond to checkpoint inhibitors, we used the ColoType CMS1-score to identify microsatellite stable (MSS) CRC tumors that have immune activity profiles similar to MSI tumors. Methods: This study was performed in Affymetrix cohort (n = 1,888) of primary CRC tumor samples assayed with hgu133plus2 Affymetrix microarrays. Degrees of infiltration of populations of immune cells were assessed with MCPcounter. Gene Set Enrichment Analysis (GSEA) using the Hallmark gene sets was used to compare biological features between subsets of samples. An 18-gene genomic score (PembroSig) of Ayers, et al, was used to predict response to Pembrolizumab. ColoType 40-gene signature identifies the CMS of a CRC tumor using values of continuous scores, one score for each of CMS1-4. CMS1+ denotes the samples with CMS1-score sufficiently high to predict it is in CMS1. A genomic score was used to predict MSI status for all samples in Affymetrix cohort; MSI predictions using said score had 97% agreement with clinically determined MSI status for 749 Affymetrix cohort samples. Results: The MSI score predicted 240 MSI samples in Affymetrix cohort, and 211 MSS samples in CMS1+ (CMS1+/MSS). The union of MSI and CMS1+/MSS compared to other samples exhibited elevated infiltration of cytotoxic lymphocytes (p < 0.0001) and CD8 T-cells (p < 0.0001), activity of interferon-gamma (p = 0.006) and interferon-alpha signaling (p = 0.012), inflammatory response (p = 0.012), and elevated PembroSig score (p < 0.0001). CMS1+/MSS compared to MSI showed no significance difference in any of these measures. None of the immune regulatory genes PD-1, PD-L1, PD-L2, CTLA4, LAG3, and IDO1 were differentially expressed between CMS1+/MSS and MSI. Of note, the only Hallmark gene sets enriched in MSI compared to CMS1+/MSS were DNA repair (p = 0.002) and MYC targets V1 (p = 0.009) and V2 (p = 0.013). We further analyzed the biological differences between MSI samples in CMS1+ (CMS1+/MSI, n = 190) and MSI samples not in CMS1+ (CMS1-/MSI, n = 50). We found that CMS1+/MSI compared to CMS1-/MSI exhibited elevated cytotoxic lymphocyte infiltration (p < 0.0001), interferon-gamma activity (p = 0.027), and PembroSig score (p < 0.0001), highlighting the relationship between immune activity and CMS1-score. Conclusions: ColoType CMS1-score identified MSS samples in Affymetrix cohort (11%) with similar immune activity as MSI samples (13%) and equivalently high values of a genomic score predictive of Pembrolizumab response.

Report From the Jerusalem Workshop on Lynch Syndrome-Hereditary Nonpolyposis Colorectal Cancer

NG03: Marine omega-3 polyunsaturated fatty acid and colorectal cancer prevention and treatment

Clearing the Air on Smoking and Colorectal Cancer

To determine the expression of estrogen receptor (ER) beta in Chinese colorectal carcinoma (CRC) patients. ERbeta expression in CRC was investigated by immunohistochemical staining of formalin-fixed, paraffin-embedded tissue sections from 40 CRCs, 10 colonic adenomas, and 10 normal colon mucosa biopsies. The percentage of positive cells was recorded, mRNA expression of ERalpha and ERbeta in 12 CRC tissues and paired normal colon tissues were detected by RT-PCR. Positive ER immunoreactivity was present in part of normal epithelium of biopsy (2/10), adenomas (3/10), and the sections of CRC tissue, most of them were nuclear positive. In CRCs, nuclear ERbeta immunoreactivity was detected in over 10% of the cancer cells in 57.5% of the cases and was always associated with cytoplasmic immunoreactivity. There was no statistical significance between ERbeta positive and negative groups in regard to depth of invasion and nodal metastases. Of the 12 CRC tissues and paired normal colon tissues, the expression rate of ERalpha mRNA in CRC tissue and corresponding normal colon tissue was 25% and 16.6%, respectively. ERbeta mRNA was expressed in 83.3% CRC tissue and 91.7% paired normal colon tissue, respectively. There was no significant difference in ERbeta mRNA level between CRC tissues and paired normal colon tissues. A large number of CRCs are positive for ERbeta, which can also be detected in normal colonic epithelia. There is a different localization of ERbeta immunoreactivity among normal colon mucosae, adenomas and CRCs. ERalpha and ERbeta mRNA can be detected both in CRC tissue and in corresponding normal colon tissue. A post-transcriptional mechanism may account for the decrease of ERbeta protein expression in CRC tissues.

A Prospective, Multicenter, Population-Based Study of BRAF Mutational Analysis for Lynch Syndrome Screening

Microsatellite Instability in Interval Colon Cancers

Colorectal carcinoma (CRC) is one of the most prevalent malignant tumors worldwide. Meanwhile, the majority of CRC related deaths results from liver metastasis. Gene expression profile of CRC patients with liver Metastasis was identified using 4 datasets. The data was analyzed using GEO2R tool. GO and KEGG pathway analysis were performed. PPI network of the DEGs between 1 and 2 gene sets was also constructed. The set 1 is named between primary CRC tissues and metastatic CRC tissues. The set 2 is named between primary CRC tissues and normal tissues. Finally, the prognostic value of hub genes was also analyzed. 35 DEGs (set 1) and 142 DEGs (set 2) were identified between CRC liver metastatic cancer patients. The PPI network was constructed using the top 10 set 1 hub genes which included AHSG, SERPINC1, FGA, F2, CP, ITIH2, APOA2, HPX, PLG, HRG and set 2 hub genes which included TIMP1, CXCL1, COL1A2, MMP1, AURKA, UBE2C, CXCL12, TOP2A, ALDH1A1 and PRKACB. Therefore, ITIH2 might represent the potential core gene for colon cancer liver metastasis. COL1A2 behaves as a key gene in colorectal carcinoma.

Introduction: Colorectal Cancer (CRC) is one of the most common malignancies worldwide. The role of MSI and KRAS somatic mutation in tumor tissue is well known in CRC. In genome-wide scale, we explored whether differential methylation is associated with MSI status. Methods: We carried out a genome-wide methylation assay (Illumina 450K) for a total of 250 paired samples from 125 CRC patients (m = 72, f = 53) at different stages (stage1:25, stage II: 33 and stageIII: 67). Of them 101 had left-sided (descending colon to rectum) CRC and 30 had MSI, and 34 had somatic mutation in KRAS (rs112445441). Results: MSI was more frequent in the right-sided tumor (54% vs. 17%, p = 0.003). Frequency of KRAS mutation was not different between right and left-sided CRC (29% vs. 27%). Among the patients with microsatellite stable (MSS) CRC (n = 95), paired comparison of methylation data between tumor and corresponding normal tissue revealed a total of 1641 tumor-specific differentially methylated loci (DML) covering 686 genes that were significant at FDR 0.001 and the magnitude of difference (delta beta) was at least 20%; Similar analysis in patients with MSI (n = 30) revealed 6209 tumor-specific DML covering 2316 genes. This suggested that MSI is associated with methylation change in much larger number of genes. We could not find any methylation signature from normal colon tissue that could predict MSI or KRAS mutation in the corresponding tumor tissue. However, we identified 413 genes to be differentially methylated in tumor tissue compared to corresponding normal tissue only in presence of MSI, irrespective of KRAS mutation status, tumor staging, and location of tumor. These are “MSI-associated tumor-specific genes”. Among these, 19 DML covering17 genes showed delta-beta >30%. The list was enriched in genes associated with biologically relevant GO-terms like, “negative regulation of cell proliferation”, “regulation of MAP kinase activity”, “regulation of biological process” etc. We also identified 240 genes differentially methylated in tumor tissue compared to corresponding normal tissue irrespective of MSI status, KRAS mutation, tumor staging, and location of tumor. These are “general tumor-specific genes”. Among these loci, 87 DML (49 genes) showed delta-beta >30%. Conclusions: Our study shows evidence of association between MSI and DNA methylation in the pathogenesis of CRC. Citation Format: Muhammad G. Kibriya, Mohammed Kamal, Mustafizur Rahman, Shantanu Roy, Maruf Raza, Rupash Paul, Habibul Ahsan, Zahidul Haq, Farzana Jasmine. Interaction of Microsatellite Instability (MSI) and tumor for Differential DNA Methylation in Colorectal carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2778.

BackgroundCircular RNAs (circRNAs) can function as sponges for microRNA (miRNA) in carcinogenesis. This study aimed to investigate the role of the circRNA of the integrin subunit alpha 5 (ITGA5) gene and microRNA-107 (miR-107) in human colorectal carcinoma (CRC) cells in vitro and tissue samples from patients with CRC and the expression of forkhead box J3 (FOXJ3) protein.Material/MethodsThirty paired CRC tissue samples and adjacent normal colon tissue samples were studied. Human CRC cell lines, including HT29, SW480, LoVo, and HIEC cells, were studied for cell proliferation using the cell counting kit-8 (CCK-8) assay. Cell migration was studied using a transwell assay, and cell apoptosis was determined using flow cytometry. The luciferase reporter assay was used to study the interactions between ITGA5 circRNA, FOXJ3, and miR-107 in human CRC cells. The expression of ITGA5 circRNA and miR-107 was determined using quantitative real-time polymerase chain reaction (qRT-PCR). The protein levels of FOXJ3 were measured by Western blot.ResultsThe expression of ITGA5 circRNA was significantly reduced in CRC tissues and CRC cell lines. High ITGA5 circRNA expression inhibited the proliferation and cell migration of CRC cells and promoted the apoptosis of CRC cells. The luciferase reporter assay confirmed that ITGA5 circRNA bound to miR-107, which directly targeted FOXJ3.ConclusionsITGA5 circRNA may act as a sponge for miR-107 to upregulate FOXJ3 expression and act as a tumor suppressor in CRC cells.

595 Background: CRCs arise through distinct mutations, including in APC pathway leading to tubular adenomas (TAs); in BRAF, with epigenetic silencing of CDX2, leading to serrated adenomas (SAs); and in the DNA mismatch repair machinery driving microsatellite instability (MSI). The APC pathway involves loss of the hormone guanylin, silencing the tumor suppressing receptor GUCY2C. Indeed, oral hormone replacement is an emerging strategy to reactivate GUCY2C and prevent CRC. Moreover, retained expression by tumors arising from TAs has established GUCY2C as a therapeutic target to prevent and treat metastatic CRC. Here, we defined the potential role of the guanylin-GUCY2C axis, and its suitability as a target, in tumors arising through the SA and MSI pathways. Methods: We compared guanylin-GUCY2C protein and mRNA expression between human TAs (n = 18), SAs (n = 15), MSI tumors (n = 7) and their matched normal adjacent tissue. Genetic mouse models of serrated and MSI tumors were used to confirm findings and elucidate mechanisms. Results: Guanylin hormone was eliminated in TAs, SAs and MSI tumors compared to their normal adjacent tissues. In contrast to the hormone, the tumor suppressing receptor GUCY2C was retained in TAs and MSI tumors. Surprisingly, GUCY2C expression was nearly eliminated in SAs reflecting loss of the transcription factor CDX2. Changes in the guanylin-GUCY2C axis in human SAs and MSI tumors were precisely recapitulated in genetic mouse models. Conclusions: Guanylin is universally lost at the earliest stages of transformation in tumors arising through divergent genomic mechanisms suggesting its utility as a biomarker of CRC initiation. These data reveal the possibility of guanylin loss silencing GUCY2C in the pathophysiology of, and oral hormone replacement to restore GUCY2C signaling to prevent, MSI tumors. Also, they highlight the potential for targeting GUCY2C to prevent and treat metastases arising from TAs and MSI tumors. In contrast, loss of GUCY2C excludes patients with SAs as candidates for GUCY2C-based prevention and therapy.

Objective To evaluate the expression of CD166 in colorectal carcinoma (CRC) tissues in order to explore its significance in CRC pathogenesis. Methods The expression of CD166 mRNA was detected by RT-PCR in CRC tissues and normal colorectal tissues outside the cancers in 23 patients with confirmed CRC. Its association with clinicopathological parameters was analyzed. Results The positive rate (95.7%) and expression level (0.309±0.145) of CD166 mRNA in CRC tissues were statistically higher than those of 39.1% and 0.109±0.255 in the normal colorectal tissues (P<0.01). No significant association of CD166 mRNA expression with age, gender, Dukes stages, tumor locations, tumor sizes or histological types of CRC was found. Conclusion CD166 mRNA expression is up-regulated in CRC tissues, which is not associated with clinicopathological parameters. Key words: Tumor stem cell; Colorectal neoplasm; CD166; Tumor marker; Dukes stage; Adenocarcinona

Colorectal carcinoma (CRC) is a common tumor in the digestive system. This study aims to elucidate the possible relationship between abnormally expressed HOXD9 and the malignant process of CRC. HOXD9 levels were analyzed in CRC and adjacent non-tumoral tissues to evaluate its prognostic value in CRC patients. Knockdown of HOXD9 was performed, and the proliferative and migratory capacities of LoVo and LS513 cells were assessed using CCK-8, transwell, and wound healing assays. Bioinformatic analysis and dual-luciferase reporter assay revealed the interaction between HOXD9 and KLK9. Rescue experiments were conducted to elucidate the co-regulation of HOXD9 and KLK9 on CRC cell behaviors. HOXD9 was upregulated in CRC tissues, and high level of HOXD9 predicted poor prognosis in CRC patients. HOXD9 was identically upregulated in CRC cell lines, especially LoVo and LS513 cells, which were used for generating HOXD9 knockdown models by transfection of sh-HOXD9. Knockdown of HOXD9 weakened proliferative and migratory capacities in CRC cells. KLK9 was the target binding HOXD9, which was downregulated in CRC tissues and cell lines. Knockdown of KLK9 reversed the inhibited proliferative and migratory capacities in CRC cells owing to HOXD9 knockdown. Highly expressed HOXD9 in CRC tissues is closely linked to the prognosis. HOXD9 stimulates CRC cells to proliferate and migrate by upregulating KLK9.

Tumor suppressor genes are often located in frequently deleted chromosomal regions of colorectal cancers (CRCs). In contrast to microsatellite stable (MSS) tumors, only few loss of heterozygosity (LOH) studies were performed in microsatellite instable (MSI) tumors, because MSI carcinomas are generally considered to be chromosomally stable and classical LOH studies are not feasible due to MSI. The single nucleotide polymorphism (SNP) array technique enables LOH studies also in MSI CRC. The aim of our study was to analyse tissue from MSI and MSS CRC for the existence of (frequently) deleted chromosomal regions and tumor suppressor genes located therein. We analyzed tissues from 32 sporadic CRCs and their corresponding normal mucosa (16 MSS and 16 MSI tumors) by means of 50K SNP array analysis. MSS tumors displayed chromosomal instability that resulted in multiple deleted (LOH) and amplified regions and led to the identification of MTUS1 (8p22) as a candidate tumor suppressor gene in this region. Although the MSI tumors were chromosomally stable, we found several copy neutral LOHs (cnLOH) in the MSI tumors; these appear to be instrumental in the inactivation of the tumor suppressor gene hMLH1 and a gene located in chromosomal region 6pter-p22. Our results suggest that in addition to classical LOH, cnLOH is an important mutational event in relation to the carcinogenesis of MSS and MSI tumors, causing the inactivation of a tumor suppressor gene without copy number alteration of the respective region; this is crucial for the development of MSI tumors and for some chromosomal regions in MSS tumors.